CN112415079B

CN112415079B - A dual-parameter self-validating homogeneous immunoassay method for single particle inductively coupled plasma mass spectrometry

Info

Publication number: CN112415079B
Application number: CN201910776155.7A
Authority: CN
Inventors: 刘睿; 黄自立; 吕弋
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-08-22
Filing date: 2019-08-22
Publication date: 2021-10-22
Anticipated expiration: 2039-08-22
Also published as: CN112415079A

Abstract

The purpose of the present invention is to provide a dual-parameter self-verified homogeneous immunoassay method, which simultaneously analyzes the frequency and intensity of a single gold nanoparticle (AuNPs) probe to realize dual-parameter detection of CEA. The principle of the present invention is: when the AuNPs probe modified with specific CEA antibody is mixed with the sample containing the CEA target, the binding of the AuNPs probe to the CEA target causes the AuNPs probe to form dimers or oligomers. This analyte-based CEA-induced aggregation of AuNPs probes resulted in a decrease in the number of probe polymers and an increase in the size of the probe polymers, resulting in changes in the frequency and intensity of aggregation of individual nanoparticles. The two-parameter measurement of CEA can be achieved by measuring the changes in the aggregation of AuNPs probes in the frequency and intensity modes of SP-ICPMS. At the same time, the mutual verification of the two modes for CEA detection can improve the reliability of sample detection.

Description

Double-parameter self-verification homogeneous immunoassay method for single-particle inductively coupled plasma mass spectrometry

Technical Field

The invention belongs to the field of detection of analytical chemistry, relates to the field of Single Particle inductive coupling Plasma Mass Spectrometry (SP-ICPMS) sensing, and particularly relates to a Carcinoembryonic Antigen (CEA) detection method based on Single Particle inductive coupling Plasma Mass Spectrometry double-parameter self-verification.

Background

Precision medicine is undergoing a rapidly growing stage and has opened a new era of medicine through research, technology and policies directed to individualized treatment. However, precision medicine often faces the dilemma of "imprecision", and it is critical that rapid, sensitive, well-defined quantification of biomolecules be performed. Homogeneous immunoassays are an attractive technique for accomplishing this task, requiring only mixing of the sample and immunochemical reagents prior to detection. The non-segregated cheapness minimizes time, laboratory consumption and automation requirements. A series of homogeneous immunoassay methods have been successfully developed and even commercialized (e.g., alphalisa by Perkinelmer and Sparcl by Lumigen), with great success in the past. Homogeneous immunoassays are one of the major analytical techniques for clinical diagnosis, and although with great success, one of the major challenges in performing homogeneous immunoassays in practical applications is the inaccuracy associated with matrix effects, since sample components are not removed by separation or washing steps. In order to ensure accurate quantification, the invention provides a self-verification uniform immunoassay method, which realizes double-parameter detection of CEA by simultaneously analyzing the frequency and the strength of a single gold nanoparticle (AuNPs) probe. Two analysis modes of the single-particle inductively coupled plasma mass spectrometry (SP-ICPMS) are well correlated, and a self-verification mechanism is provided for accurate immunoassay of CEA. Both modes provide a linear range of two orders of magnitude and a limit of detection (LOD) at the pmol level. Due to the self-verification strategy and the high tolerance of ICPMS to the matrix effect, the method of the invention is successfully applied to the immunodetection of a series of human serum samples, and the result is consistent with the clinical routine method.

Disclosure of Invention

The invention aims to provide a self-verification homogeneous immunoassay method based on single particle accurate counting and application thereof in carcinoembryonic antigen detection. The principle of the invention is as follows: the method is based on analyte-induced agglomeration of AuNPs probes. When AuNPs probes modified with specific CEA antibodies are mixed with a sample containing CEA targets, the binding of AuNPs probes to CEA targets results in the AuNPs probes forming dimers or oligomers. This binding-induced aggregation of AuNPs probes results in a decrease in the number of probe polymers and an increase in the AuNPs probe polymer size, thereby causing changes in the frequency and intensity of aggregation of individual nanoparticles. The double-parameter measurement of the CEA can be realized by measuring the aggregation change of the AuNPs probe through two modes of SP-ICPMS frequency and intensity. The mutual verification of the two modes for CEA detection can also improve the reliability of sample detection.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention firstly prepares the AuNPs probe modified by the specificity CEA antibody. The principle is as follows: CEA antibody was added to a 30 nm AuNPs solution buffered with boric acid (pH 9.0) to make it weakly alkaline (pH 8.0) by the principle of electrostatic adsorption. Under the condition of pH about 8.0, the antibody is not charged at the isoelectric point, and can be adsorbed to the surfaces of AuNPs wrapped by negative electricity in an electrostatic adsorption mode. And then Bovine Serum Albumin (BSA) is added to block unadsorbed sites on the surfaces of the AuNPs so as to prevent nonspecific adsorption.

The two-parameter detection process of CEA is as follows: the reaction time of the sample with the AuNPs probe was determined to be 37 after a series of optimizations^oC, incubating for 60 min at the reaction temperature; before detection, 10000 times of samples need to be diluted, and then SP-ICPMS double-parameter time resolution detection is carried out on the diluted solution; and in the SP-ICPMS frequency mode, the diluted sample solution is subjected to time resolution signal acquisition, the acquisition time is 60 s, and the acquisition rate is 50 mu s. Under the SP-ICPMS intensity mode, carrying out time resolution signal acquisition on the diluted sample solution, wherein the acquisition time is 60 s, and the acquisition rate is 50 mus/time; wherein the linear range of CEA sample detection is 0.78 ng/mL to 100 ng/mL (frequency mode) and 1.25 ng/mL to 100 ng/mL (intensity mode); the entire course of the detection of the samples was carried out in PBS buffer (0.01M pH 7.4, containing 137 mM NaCl and 2.7 mM KCl solution); the detection limit of the method of the invention can reach 0.21 ng/mL (about 1.2 pM) and 0.68 ng/mL (about 3.8 pM) respectively in the frequency mode and the intensity mode.

The invention has the following advantages: the invention provides a self-verification double-parameter immunoassay method, which carries out quantitative detection on CEA by accurately counting a single AuNPs probe and analyzing the particle size through SP-ICPMS. In the presence of different concentrations of analyte, the AuNPs probes aggregated into oligomers to varying degrees. The pulse frequency information of SP-ICPMS reveals the number change of the AuNPs probes, and the pulse intensity information shows the size change of the AuNPs probe aggregates. By monitoring both modes simultaneously, both modes provide a linear range of two orders of magnitude, accompanied by a detection limit for the pM level. Due to the self-verification advantages of the two modes and the high tolerance of ICPMS, the homogeneous immunoassay provided by the method is successfully applied to human serum samples, and the great potential of the method in clinical diagnosis is reflected.

Drawings

The invention adopts the double-parameter analysis performance research of AuNPs probe group poly-p-CEA based on the induction of the analyte.

FIG. 1 is a diagram showing the mechanism and phenomenon demonstration of the analysis method of the present invention

FIG. 2 is a chart of the feasibility study of two quantitative patterns in the assay of the invention

FIG. 3 is a schematic diagram showing the analytical performance of the analytical method of the present invention in the frequency mode and the intensity detection mode

FIG. 4 is a graph showing the results of two-parameter self-verification of actual blood samples according to the analysis method of the present invention

FIG. 5 is a graph showing the spiked recovery exploration results of blood samples according to the assay method of the present invention.

Detailed description of the preferred embodiments

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The water used in the following examples was ultrapure water, which was treated with a Milli-Q ultrapure water purification system. All samples in the following examples were not purified prior to use.

The invention is carried out according to the following specific steps:

homogeneous immunoreaction:

preparation of a.30 nm AuNPs

a1 adding 1 mL of 1% chloroauric acid (w/v) into 90 mL of ultrapure water, placing magnetons into a three-necked bottle, mixing uniformly, heating in a water bath under reflux for 5 min,

a2 adding 1.7 mL of 1% sodium citrate reducer, stirring and maintaining reflux for 20 min,

a3, closing the water bath, keeping the stirring of the stirrer, slowly cooling the solution to room temperature, pouring the solution into a 100 mL storage bottle, and fixing the volume to 100 mL;

antibody modification of 30 nm AuNPs

b1 adding 100 μ L of pH 9.0 boric acid buffer solution into 1 mL of AuNPs, adjusting pH to about 8.0-8.4 to make IgG protein near isoelectric point (no self-charge), mixing by vortex,

b2 adding 30 μ g CEA antibody, mixing by vortex, reacting at room temperature (25 deg.C) for 30 min,

b3 adding blocking agent 10% BSA solution 150 uL, blocking at room temperature (25 deg.C) for 30 min,

b4 solution after reaction at 4^oCentrifuging at 8000-;

c. immune response

c1 adding 100 μ L10 times diluted AuNPs-CEA antibody biological probe into 500 μ L centrifuge tube, then adding 30 μ L serum sample, finally adding 100 μ L10 times diluted AuNPs-CEA antibody biological probe,

after the c2 solution was vortexed uniformly, it was vortexed at 37^oC, incubating for 60 min;

d. sample dilution

d1 sucking 20 μ L of reacted solution, adding into 2 mL centrifuge tube, adding 2 mL ultrapure water, mixing by vortex,

d2 adding 40 μ L of the diluted solution into a 4 mL centrifuge tube, adding 4 mL of ultrapure water, and mixing by vortex;

SP-ICPMS frequency mode determination

e1 the pipette of the ICPMS was inserted into the diluted solution,

e2 setting frequency mode to collect signal of solution at speed of 50 μ s/time for 60 s;

SP-ICPMS Strength Pattern determination

f1 the pipette of the ICPMS was inserted into the diluted solution,

f2 setting an intensity mode to collect signals of the solution, wherein the collection time is 60 s, and the speed is 50 mus/time;

the following description will be further described with reference to the drawings attached to the specification, but the analysis method of the present invention is not limited to the following examples.

Example 1 investigation of whether analyte-induced aggregation of AuNPs probes would lead to a change in SP-ICPMS Signal

To investigate whether the aggregation of AuNPs probe induced by CEA can cause signal change in SP-ICPMS, based on the antigen-antibody reaction of CEA, the real-time signal change of the reaction sample in SP-ICPMS in the presence of different concentrations of CEA by AuNPs probe was investigated. In the experiment, CEA with different concentrations is added into AuNPs probe solution, and the solution after reaction is analyzed by SP-ICPMS through dilution. As can be seen in FIG. 1, the real-time signal appears to be of relatively low intensity and relatively stable and uniform signal in the absence of the CEA sample. With the increasing CEA concentration, the real-time signal has the tendency of increasing intensity, and the number of the signals also has the tendency of decreasing with the aggregation of the AuNPs probe. In the presence of CEA at a concentration of 100 ng/mL, a significant increase in real-time signal and a reduced amount of signal was observed. Therefore, the embodiment fully proves that the antigen-antibody recognition system established by the method can cause the change of SP-ICPMS signals in the presence of CEA, and the two-parameter quantitative detection of CEA can be realized by using the valuable signal changes.

Example 2 feasibility study of two modes in the assay method of the invention

Because the analysis method adopts a double-parameter self-verification mechanism, before the analysis method is applied to homogeneous immunoassay, feasibility analysis needs to be carried out on two modes;

a. feasibility study on frequency mode: in the frequency mode, the frequency information analyzed by the instrument can reflect the number of the AuNPs probe aggregates in the sample. And (3) continuously diluting the newly-prepared AuNPs probe by a certain multiple to obtain probe solutions with a series of concentrations. After dilution by a certain factor, AuNPs probe solutions of different concentrations were analyzed in a frequency mode SP-ICPMS with a scanning time of 60 s and a speed of 50. mu.s/time. The results are shown in FIG. 2, where the concentrations of AuNPs probes were from 0 to 5.11X 10⁵The number of the corresponding SP-ICPMS frequencies is increased correspondingly per mL, and the change is in a linear relation with the change of the concentration of the changed AuNPs, and the correlation coefficient is 0.9991. Therefore, under the condition of CEA, the AuNPs probe can agglomerate to different degrees, and the CEA concentration can be researched through the change of the number of the AuNPs probe agglomerates before and after the reaction;

b. investigation of the feasibility of the intensity pattern: in the intensity mode, the intensity information analyzed by the instrument can reflect the size of the AuNPs probe aggregate in the sample. A series of AuNPs sample solutions with different particle sizes are prepared to simulate AuNPs probe aggregates with different particle sizes, wherein the particle sizes of the AuNPs probe aggregates are 21 nm, 30 nm, 34 nm, 43 nm and 78 nm respectively. After dilution by a certain factor, the concentrations of AuNPs sample solutions of the respective particle sizes were kept equivalent. AuNPs probe solutions of different particle sizes were analyzed in the intensity mode SP-ICPMS with a scan time of 60 s and a speed of 50. mu.s/time. As shown in FIG. 2, the corresponding SP-ICPMS signal intensity increases with the increasing particle size of the AuNPs solution, and the change of the intensity is logarithmically linear with the changed particle size of the AuNPs, and the correlation coefficient is 0.9999. Therefore, the AuNPs probe can agglomerate in different degrees under the condition of CEA, and the CEA concentration can be researched through the change of the particle size of the AuNPs probe agglomerate before and after the reaction.

Example 3 investigation of the linearity of detection of CEA by the assay of the invention

The homogeneous immunoassay of CEA based on SP-ICPMS is explored in the embodiment, and the linearity of two modes in the established two-parameter self-verification mechanism is respectively explored;

a. linearity of frequency pattern versus CEA detection: in the frequency mode, after the sample solution after the reaction is diluted 10000 times, in the SP-ICPMS, the time resolution signal collection is carried out by the frequency mode, the collection time is 60 s, and the collection speed is 50 mus/time. As shown in FIG. 3, a linear relationship of 0.78-100 ng/mL was obtained according to the proportional relationship between the CEA concentration and the frequency response signal, and the linear correlation coefficient was 0.9991. Calculating the detection limit of 0.21 ng/mL by linear regression analysis;

b. linearity of intensity pattern versus CEA detection: in the intensity mode, after the reaction-completed sample solution was diluted 10000 times, time-resolved signal acquisition was performed in the intensity mode at an acquisition time of 60 s and an acquisition rate of 50. mu.s/time in SP-ICPMS. As shown in FIG. 3, a linear relationship of 1.25-100 ng/mL was obtained according to the proportional relationship between the CEA concentration and the intensity response signal, and the linear correlation coefficient was 0.9993. For linear regression analysis, a detection limit of 0.68 ng/mL was calculated.

Example 4 investigation of CEA Selectivity of the assay of the invention, correlation of the two modes

a. Selectivity of the method of the invention to CEA: the method adopts 100 ng/mL as the concentration of CEA sample in the interference experiment, and the adopted interference antigen and the concentration are respectively 1 mug/mL of Alpha Fetoprotein (AFP), 1 mug/mL of human immunoglobulin G (IgG) and 500U/mL of carbohydrate antigen 199 (CA 199). The method also explores the influence of certain salinity conditions on a reaction system, and explores by taking a 10 mg/mL NaCl solution as salinity. According to the experimental steps, CEA and an interfering substance are mixed in sequence, and after complete reaction with an AuNPs probe, a sample is diluted by 10000 times, and the sample is analyzed in a strength mode under SP-ICPMS. The result is shown in fig. 4, except that CEA causes AuNPs probe specific recognition and aggregation, resulting in obvious intensity information change, the rest interfering substances are equivalent to blank signals, and the specificity of the established homogeneous immunization method to CEA and the tolerance to salinity are considerable;

b. the correlation between the two modes in the method of the invention: the method adopts a frequency mode and an intensity mode to detect the CEA concentration in the human serum sample, and the obtained results are calculated according to the linearity obtained by the two modes respectively to obtain the CEA concentration value. Finally, the concentration values obtained from the two modes are compared with each other. As shown in FIG. 4, the CEA concentration in the serum was in the range of 0 to 80 ng/mL, the CEA concentration measured in the frequency mode and the CEA concentration measured in the intensity mode were well matched to each other, and the correlation coefficient between the two modes was 0.9875.

Example 5 investigation of the assay method of the invention detection and spiking recovery of CEA in actual blood samples

a. Collecting serum: the serum adopted in the application of the analysis method is from the seven hospitals in metropolis;

b. blood sample detection: the sample volume of each serum sample was 30 uL, and the CEA content in each serum was determined by ELISA kit (purchased from Shanghai Touchi Biotech Co., Ltd.) before the assay method of the present invention was used. The analytical procedure for the detection was in accordance with the above. The results are shown in FIG. 4, the CEA content measured by SP-ICPMS is in good agreement with the value obtained by ELISA in the CEA detection linear range of the method of the invention, and the correlation coefficient of the two methods is 0.9948;

c. adding a standard and recovering: group 12 serum samples were divided into 2 groups, wherein one group of 6 serum samples were mixed with an equal volume (100. mu.L) of 100 ng/mL CEA standard, and the other group of 6 serum samples were mixed with an equal volume (100. mu.L) of 50 ng/mL CEA standard. The resulting sample solution was investigated according to the above-mentioned research procedure and tested with SP-ICPMS. The results are shown in fig. 5, the established method can obtain the recovery rate of 93-116%, and the analysis method of the invention is proved to have the analysis and detection capability of actual samples.

Claims

1. A double-parameter self-verification homogeneous immunoassay method of single-particle inductively coupled plasma mass spectrometry is characterized in that: the analysis method comprises a CEA antibody functionalized gold nano (AuNPs) probe, in the presence of CEA, the CEA can be quantitatively detected through antigen-antibody specific recognition, CEA-induced AuNPs probe aggregation, and single-particle inductively coupled plasma mass spectrometry (SP-ICPMS); the system was carried out in PBS buffer solution (0.01M pH 7.4, containing 137 mM NaCl and 2.7 mM KCl) at pH 7.4; the method of the invention modifies AuNPs by adding 30 mug CEA antibody into each 1 mL AuNPs solution in an electrostatic adsorption mode, and the centrifugal condition of probe purification is 4^oCentrifuging at 8000-11000 rpm for 25 min under C, modifying AuNPs probe of CEA specific antibody, generating antigen-antibody specific recognition for the sample with CEA, leading the AuNPs probe induced by CEA of analyte to aggregate, the system reaction time is 60 min, the temperature is 37^oC; two modes of inductively coupled plasma mass spectrometry (SP-ICPMS) by single particle: the frequency mode and the intensity mode are used for analyzing the aggregate of the AuNPs probe induced by the analyte CEA to achieve the aim of quantitatively detecting the CEA, and the exploration of the feasibility of the frequency mode is realized by measuring the frequency of 30 nm AuNPs probes with different concentrations; the exploration on the feasibility of the intensity pattern is generalThe method is realized by measuring the strength of Au in AuNPs solutions with different particle sizes; before immunoassay, the dilution multiple of a sample is 10000 times, the time of frequency mode measurement in SP-ICPMS is 60 s, and the scanning rate is 50 mus/time; the time for intensity pattern determination was 60 s and the scan rate was 50. mu.s/time.

2. The assay as recited in claim 1 wherein a two parameter assay for analyte CEA is achieved by combining the frequency and intensity modes of SP-ICPMS, the frequency mode having a linear range of 0.78 ng/mL to 100 ng/mL for CEA determination and a detection limit of 0.21 ng/mL; the linear range of intensity pattern to CEA determination is 1.25 ng/mL to 100 ng/mL with a detection limit of 0.68 ng/mL.